产酶溶杆菌中一个独特的c-di-GMP代谢基因lchR调控抗菌物质HSAF合成的机制研究

Lysobacter enzymogenes is an important Gram-negative bacterial biological control agent. This bacterium produces an antifungal metabolite, HSAF, which has a new structure, a new mode of action and a unique biosynthetic mechanism, but has a low toxicity. Unfortunately, the yield of HSAF in wild-type strain is very low even under a HSAF-inducible condition, which is probably due to the precise and intricate regulation of HSAF biosynthesis by a diverse array of regulators. The low yield limits the application of HASF as a new bio-fungicide. Cyclic di-GMP (c-di-GMP), a new bacterial second messenger, exerts its important biological functions through interaction with specific receptors in its mediated pathways in bacterial cells. However, little is known about the function and regulation of c-di-GMP in the biosynthesis of secondary metabolites in any bacterial biological control agent. Recently, we identified a c-di-GMP metabolism gene (lchR) with multiple domains and ability in positive regulation of HSAF biosynthesis in L. enzymogenes. Importantly, this gene appears to be uniquely distributed in the genome of diverse Lysobacter species, suggesting the regulatory effect of lchR-mediated c-di-GMP pathway on antimicrobial secondary-metabolite biosynthesis (e.g. HSAF biosynthesis) is unique and probably a new mechanism in Lysobacter species. The goal of this project is to figure out the role of the lchR and its different domains in the biosynthesis of HSAF, and in the metabolism (synthesis or degradation) of c-di-GMP in L. enzymogenes. We also plan to identify the specific receptor (transcriptional factor) of c-di-GMP in the lchR-mediated pathway, and to explore the mechanism of interaction between this specific receptor and c-di-GMP, as well as to understand the regulatory pattern of this receptor on HSAF biosynthesis at biochemical and molecular level in L. enzymogenes. The outcomes of the project are expected to reveal a new c-di-GMP-mediated regulatory mechanism in Lysobacter, or even in bacterial level.

产酶溶杆菌是一种重要的生防细菌。HSAF是该菌中新报道的一种毒性低、结构新颖、作用方式和生物合成途径独特的广谱抗真菌卵菌代谢产物。c-di-GMP是细菌中一种新型第二信使，在细胞内它通过与特异的受体互作来行使调控功能，但其在生防细菌中调控抗菌物质生物合成方面的研究鲜有报道。申请者近期从产酶溶杆菌中鉴定了一个多结构域并调控HSAF合成的c-di-GMP代谢基因lchR，并发现该基因在非溶杆菌属细菌中没有同系物，揭示其介导的c-di-GMP信号途径在产酶溶杆菌中可能是通过一种独特机制来调控HSAF合成的。本项目将解析lchR及其各结构域在HSAF合成及c-di-GMP代谢(合成或降解)中的功能；鉴定lchR作用途径中的c-di-GMP受体转录因子，阐明该受体转录因子与c-di-GMP的结合机理及其调控HSAF合成的机制。研究结果有望在溶杆菌中揭示c-di-GMP行使调控功能的一种新机制。

产酶溶杆菌是一种重要的生防细菌。HSAF是该菌中新报道的一种毒性低、结构新颖、作用方式和生物合成途径独特的广谱抗真菌卵菌代谢产物。c-di-GMP是细菌中一种新型第二信使，在细胞内它通过与特异的受体互作来行使调控功能，但其在生防细菌中调控抗菌物质生物合成方面的研究鲜有报道。本项目从产酶溶杆菌中鉴定了一个多结构域并调控HSAF合成的c-di-GMP代谢基因lchR，并发现该基因在非溶杆菌属细菌中没有同系物，揭示其介导的c-di-GMP信号途径在产酶溶杆菌中可能是通过一种独特机制来调控HSAF合成的。进一步发现LchP是一个既含有GGDEF结构域（负责合成c-di-GMP）又含有EAL结构域（负责降解c-di-GMP），同时在其N端拥有3种不同的信号感应结构域的蛋白。转录组数据揭示LchP蛋白能够特异性调控HSAF的生物合成。虽然LchP通过其降解酶活性来调控HSAF的生物合成，但其负责合成c-di-GMP的GGDEF酶活关键位点在这个过程中也至关重要。进一步研究鉴定了一个位于LchP下游的c-di-GMP的受体转录因子Clp。Clp与LchP发生特异性物理物质。这种互作有利于Clp促进LchP的降解酶活性。体内c-di-GMP浓度升高，c-di-GMP与Clp形成复合物，降低Clp与HSAF合成关键基因lafB启动子区的结合，从而最终抑制HSAF的生物合成。本研究阐明了LchP与c-di-GMP的受体Clp互作是实现c-di-GMP对HSAF特异性调控的一种新途径，据此揭示了c-di-GMP代谢酶与其受体互作是c-di-GMP实现信号途径特异性传导的一种新机制。通过对该机制的分子改良，我们构建了HSAF的高产菌株，推动了HSAF的基础研究新发现向应用研究的转化进程。研究结果共发表SCI论文7篇。结果发表后，mBio编辑评价认为该机制（即c-di-GMP受体蛋白与其降解酶互作）是细菌c-di-GMP信号途径特异性传导的一个新发现。同时应Science Signaling编辑邀请，撰写综述。

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